Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force

Abstract

Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

Fig. 1. Electron microscopy of cell-cell junctions and optical trap-based assay setup

(A) Transmission electron microscopy (TEM) image of a cell-cell junction between Caco-2 epithelial cells cultured on EM-amenable substrates. Scale bar is 1 μm. Brackets label actin filament arrays parallel to the junction, and the yellow arrow marks where the actin arrays were in close proximity to cell-cell contacts. (B) Three-dimensional electron tomography reconstruction of the same region shown in (A) rotated 90° clockwise and then tilted 45° around the horizontal axis. The cell-cell junction is highlighted in red. Yellow arrow marks the same region as in (A). Scale bar is 1 μm. (C, D) Fluorescence time-lapse micrographs of tetramethylrhodamine (TMR)-labeled F-actin (white lines) at the surface of a coverslip coated with either 1 μM Mm cadherin-catenin complex (C) or 2 μM Mm αE-catenin homodimer (D). Insets in solid yellow lines show the corresponding images for regions bounded in the dashed yellow lines after 30 s had elapsed; actin filaments remained stably bound to coverslips coated with Mm αE-catenin homodimer, whereas actin filaments diffused away from coverslips coated with the Mm cadherin-catenin complex within seconds. Out-of-focus features are glass microsphere platforms (see below). Scale bars are 10 μm. (E) Illustration of a cadherin-catenin complex and actin filament reconstituted in the optical trap assay (not to scale). GFP-E-cadherin cytoplasmic tail (green), β-catenin (yellow) and αE-catenin (blue) are immobilized on a coverslip. Glass microspheres (1.5 μm-diameter) on the coverslip act as platforms such that cadherin-catenin complexes can contact the actin filament. A single biotinylated, TMR-phalloidin-coated actin filament (red) extends between two 1 μm-diameter streptavidin-coated beads held in optical traps (pink). (F) Top view of the assay in bright field. Beads attached to the actin filament (not visible) are held in traps 1 and 2, and the platform bead is positioned between the traps and below the filament. Scale bar is 2 μm.

Fig. 2. Mm cadherin-catenin complexes bound actin filaments in oscillating-stage experiments

(A) The illustrations show that upon binding, the motion of the stage was transmitted to the trapped beads, and the force exerted on them was correlated with the motion of the stage, as shown in the time series in (B). The trapped beads stopped moving along with the stage when the surface-bound cadherin-catenin complex detached from the actin filament. (B) Part of a representative time series of force exerted on one of the two optically trapped beads attached to an actin filament (blue). ~1 μM Mm cadherin-catenin complex was purified and added to the flow cell. The stage was driven by a 150 nm-amplitude, 50 Hz-frequency sine wave (gray). (C) Full force time series from which the binding events in (B) came (shaded in teal). Peaks in the series are individual binding events, most of which lasted for approximately one half period of the sine wave used to drive the stage. (D) Force time series from a negative control experiment in which ~1 μM Mm E-cadherin/β-catenin complex was purified without Mm αE-catenin and added to the flow cell. Oscillation amplitude and frequency of the sine wave used to drive stage were the same as in (B). (E) Frequency of observed Mm cadherin-catenin complex/F-actin binding as a function of maximum stage speed (amplitude × angular frequency); n = 297, bin width is 10⁴ nm/s. Event frequency is the number of binding events divided by the total time sampled. (F) Sine histogram of the number of Mm cadherin-catenin complex/F-actin binding events which started in each angle bin (legend shows counts; n = 235, bin width is 36°, binding events from trap 1 are in blue and those from trap 2 are in green). All events were from the 4×10⁴ nm/s-bin shown in (E). The glass microsphere platform (A) was farthest from trap 1 when it was at +1, and from trap 2 when the stage position was at -1. Stage position was normalized by the maximum amplitude of the wave.

Fig. 3. Measurement of Dr cadherin-catenin complex/F-actin bond lifetimes in constant-stage-position experiments

(A) An actin filament binding event representative of those observed in the presence of cadherin-catenin complexes reconstituted with 10 nM of added Dr αE-catenin (see text). The stage was driven using a wave in the shape of a trapezoid with a 100-nm height and 1×10⁴ nm/s slope (top: force time series in blue; bottom: stage position time series in black). Piecewise-constant fit of the force signal is shown in red, and the black arrow points to the segment whose duration and magnitude are the lifetime of the last Dr cadherin-catenin/F-actin bond and the force exerted on it, respectively. The black bar underneath the trace represents the total bound time of the entire event. (B) A representative force time series with the event in (A) shaded in teal. (C) Representative single-molecule force time series showing binding between a surface-bound Dr cadherin-catenin complex and an actin filament in the presence of 100 nM Mm αE-catenin ABD. Flow cell was prepared using 1 nM added Dr αE-catenin. (D) Force time series showing an actin-binding event measured for multiple surface-bound Dr cadherin-catenin complexes and 100 nM Mm ABD. The black bar above the trace represents the total bound time of the entire event. Flow cell was prepared using 5 nM added Dr αE-catenin. (E) Survival frequency of total bound times, as marked by the black bar in (A) and (D), measured in experiments using flow cells prepared with 5 nM added Dr cadherin-catenin complex (red: no Mm ABD present, and n = 412; blue: 100 nM Mm ABD, and n = 107). The survival frequency at time t is the fraction of complexes that remain bound for durations greater than t.

Fig. 4. Force-lifetime distribution of Dr cadherin-catenin complex/F-actin bonds and lifetime survival analysis

(A) Two-dimensional histogram of Dr cadherin-catenin complex/F-actin bond force-lifetime values measured from the last piecewise constant segment in multi-step unbinding events, as shown in Fig. 3A. Tick labels on color bar are bin counts (17 force bins, 32 lifetime bins, and n = 803). (B) Kinetic schemes representing dissociation from either one bound state (blue) or two bound states (red). (C) Survival frequencies of Dr cadherin-catenin complex/F-actin bond lifetimes from three force bins indicated in Fig. 4A (black arrows, n = 188 in bin F₁, 185 in F₂, and 129 in F₃; errors are SEM). Red lines are least-square fits of a bi-exponential function (two bound states), and blue lines are those of a single exponential function (one bound state). R² > 0.90 for the bi-exponential function in all force bins, and the additional parameters of the bi-exponential function are justified (p ~0 in F-test). Additional force bins are shown in fig. S2A.

Fig. 5. Two-state catch bond model

(A) Kinetic schematics of the 2-bound state models that were considered. In a two-state catch bond model (left), states 1 and 2 are weakly and strongly bound states, respectively. State 0 is the unbound state. Unbinding rates k₁₀ and k₂₀ increase exponentially with respect to force. Transitions between states 1 and 2 occur at rates k₁₂ and k₂₁. The transition rate k₁₂ increases exponentially with force, while k₂₁ does the opposite. These transitions do not occur in the independently bound states model (right). (B) Averages of Dr cadherin-catenin complex/F-actin bond lifetimes binned by force (black dots, error bars are SEM, and n = 803). The red curves show the mean lifetime distributions predicted by the two-state catch bond model (solid red) and a two independently bound states model (dashed red). Model parameters were computed using maximum likelihood estimation. (C) Survival frequencies of Dr cadherin-catenin complex/F-actin bond lifetimes from Fig. 4C, compared with survival probability curves predicted by the two-state catch bond model (red lines, R² > 0.90 for all bins except for the 4 pN bin, R² = 0.67). (D) Two-state catch bond model dissociation rates as functions of force. (E) Table of maximum likelihood-estimated parameters of the two-state catch bond model and their 95% confidence intervals determined by parametric bootstrapping.